Extended Data Fig. 7: Proteomic and m6Am-seq analysis identifies Pcif1 downstream targets.
a, Metabolic labeling analysis to measure total protein synthesis in both WT and Pcif1 KO CD8+ T cells. b, c, GSEA shows enrichment of ferroptosis (b) and T cell-mediated cytotoxicity (c) pathways in Pcif1 KO CD8+ T cells. NES: normalized enrichment score. d, m6Am levels of naïve CD8+ T cells were quantified by LC-MS/MS. n = 2 biological replicates with 3 technical replicates each. e, m6Am-seq workflow. Total RNA was fragmented and immunoprecipitated with a cap-m7G antibody (m7G-IP), followed by [FTO (+)] or [FTO (−)] treatment, then m6A-IP. Sequencing profiles were normalized and compared, identifying ‘Demethylase-sensitive peaks’ as m6Am. f, Correlation analysis of m6Am enrichments between replicates. g, Metaplot showing m6Am overlapped with CAGE-annotated TSSs. h, Relative mRNA levels of the top 10 genes in activated WT and Pcif1 KO CD8+ T cells. n = 3 mice/each group. i, Genome browser views of Fth1, Cd69 and Slc3a2 using m6Am-seq: The 5′-UTR methylation peak decreased in ‘FTO (+)’ vs. ‘FTO (-)’ samples, indicating a demethylase-sensitive m6Am (upper panel). High-SRD adenosine residues were defined as m6Am sites, overlapping with FANTOM TSS (lower). j, WB quantification of Fth1, Cd69, and Slc3a2 in splenic activated WT or Pcif1 KO CD8+ T cells. n = 3 mice/each group. k-m, RNA expression of target genes at specific time points. n = 3 mice/each group. n-p, RNA decay assay showing Fth1, Slc3a2, Cd69 mRNA level in activated CD8+ T cells post-actinomycin D treatment. n = 3 mice/each group. q, WB analysis of Ctbp2 knockdown on target genes expression in WT and Pcif1 KO CD8+ T cells. r, GO analysis of PCIF1-interacting proteins (log2 FC > 2) highlights top 10 enriched pathways. s, 7-AAD staining of activated CD8+ T cells, treated for 24 hours with 0.1 μM RSL3 or 20 μM Erastin. n = 3 mice/each group. Each symbol represents an individual mouse. One-sided permutation test (b,c), one-sided hypergeometric test, without adjustment (r), two-way ANOVA followed by Sidak’s multiple comparisons test (k-p,s) or two-tailed unpaired Student’s t-test (d,i,j) were performed. Data are shown as the mean ± SEM.
